A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors

Background and Purpose Helix stapling enhances the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cytoplasmic targeting. We scrutinized to which degree the pharmacokinetic characteristics are a function of the staple and differ from those of the standard cationic cell-penetrating peptide nonaarginine for a peptide inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein. Experimental Approach Stapled peptides and linear counterparts were synthesized in the free and fluorescently-labeled form. Activity was determined in biochemical time-resolved Forster resonance energy transfer experiments and cellular high-content assays. Cellular uptake and intracellular trafficking were visualized by confocal microscopy. Key Results Peptides showed sub-nanomolar potency. For short-time incubation, uptake efficiencies for the stapled and linear peptides were similar and both were taken up less efficiently than nonaarginine. Only for SJSA-1 cells expressing the Hdm2 target protein the stapled peptides showed an enhanced cytoplasmic and nuclear accumulation after long-term incubation. This was also observed for the linear counterparts, albeit to a lesser degree. For HeLa cells, which lack target expression, no such accumulation was observed. Conclusion and Implications The cytosolic and nuclear accumulation are not an intrinsic property of the stapled peptide, but result from capture by the target Hdm2 after endo-lysosomal release. Considering the rather poor upake, the further development should focus on an increase of uptake efficiency.